7700–7713 Nucleic Acids Research, 2016, Vol. 44, No. 16
doi: 10.1093/nar/gkw494
Published online 1 June 2016
MutS regulates access of the error-prone DNA
polymerase Pol IV to replication sites: a novel
mechanism for maintaining replication fidelity
Lucı́a M. Margara1 , Marisa M. Fernández2 , Emilio L. Malchiodi2 , Carlos E. Argaraña1 and
Mariela R. Monti1,*
1
Centro de Investigaciones en Quı́mica Biológica de Córdoba (CIQUIBIC), CONICET, Departamento de Quı́mica
Biológica, Facultad de Ciencias Quı́micas, Universidad Nacional de Córdoba, Ciudad Universitaria, Córdoba
X5000HUA, Argentina and 2 Cátedra de Inmunologı́a and Instituto de Estudios de la Inmunidad Humoral Profesor
Ricardo A. Margni, CONICET, Facultad de Farmacia y Bioquı́mica, Universidad de Buenos Aires, Buenos Aires
C1113AAD, Argentina
Received December 17, 2015; Revised May 4, 2016; Accepted May 20, 2016
ABSTRACT
Translesion DNA polymerases (Pol) function in the
bypass of template lesions to relieve stalled replication forks but also display potentially deleterious
mutagenic phenotypes that contribute to antibiotic
resistance in bacteria and lead to human disease.
Effective activity of these enzymes requires association with ring-shaped processivity factors, which
dictate their access to sites of DNA synthesis. Here,
we show for the first time that the mismatch repair
protein MutS plays a role in regulating access of the
conserved Y-family Pol IV to replication sites. Our
biochemical data reveals that MutS inhibits the interaction of Pol IV with the ␤ clamp processivity factor
by competing for binding to the ring. Moreover, the
MutS–␤ clamp association is critical for controlling
Pol IV mutagenic replication under normal growth
conditions. Thus, our findings reveal important insights into a non-canonical function of MutS in the
regulation of a replication activity.
INTRODUCTION
Replication of chromosomal DNA is a highly organized
process performed by multi-protein replisome complexes.
One of the main replisome components is the replicative DNA polymerase (Pol) that concurrently synthesizes a
high-fidelity copy of the leading and lagging strands (1). Pol
III holoenzyme (HE) is responsible for the bulk of chromosomal replication in bacteria, whereas the eukaryotic replisome involves Pol ⑀ and Pol ␦. Replisomes are likely to encounter lesions on the template that generally inhibit DNA
synthesis by replicative Pols and are believed to impede fork
* To
progression, which constitutes a potentially lethal event (2).
Most organisms have evolved to possess specialized Pols
that efficiently incorporate nucleotides opposite and past
DNA lesions in a process known as translesion synthesis
(TLS) (1,3). Escherichia coli possess three distinct TLS enzymes: Pol II, Pol IV and Pol V. In mammalian cells, Pol ␨ ,
Pol ␩, Pol ␬ and Pol ␫, and possibly Pol ␪ and Pol ␯ play a
role in TLS. Genetic and biochemical analysis demonstrates
that most alternative Pols bypass template lesions in either
a mutagenic or an error-free manner, depending on the nature of the lesion and the sequence context, but exhibit low
fidelity of synthesis when copying undamaged DNA. Thus,
although the activities of specialized Pols promote cell survival by allowing the release of replication blocks, their access to sites of DNA synthesis must be tightly controlled to
avoid extensive mutagenesis or other potentially deleterious
effects on the cell.
Pol IV function has been extensively studied in recent
years. This TLS Pol is conserved among all life domains (4)
and participates in diverse biological transactions (3). Pol
IV catalyzes accurate TLS over N2 -dG adducts and alkylation lesions and the error-prone bypass of oxidized bases (5–
7). It has been postulated that Pol IV may also relieve stalled
replication forks on undamaged DNA by extending terminal mismatches created but unable to be extended by Pol
III HE, or by assisting Pol III HE during synthesis across
difficult sequence contexts (8,9). Pol IV shows a high basal
concentration in exponentially growing cells compared to
expression levels of Pol III HE and other TLS Pols (1,3).
However, access of Pol IV to replication sites is limited during normal growth, as suggested by the fact that basal levels of Pol IV do not significantly contribute to spontaneous
mutagenesis (5,10). Pol IV expression is upregulated under
stressful conditions, thus contributing to mutagenesis that
could ultimately result in antibiotic resistance and adapta-
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Nucleic Acids Research, 2016, Vol. 44, No. 16 7701
tion of pathogenic bacteria (5,11–12). Notably, an improved
understanding of mechanisms implicated in the regulation
of Pol IV access to replication sites will help to develop new
therapies to control its function in the acquisition of drug
resistance and bacterial pathogenic process.
Multiple mechanisms have been proposed to function in the coordinate regulation of access to DNA
replication sites of specialized Pols (1). One mechanism
that has gained considerable attention refers to the role
played by sliding clamps, the prokaryotic ␤ clamp and
the archaeal/eukaryotic proliferating cell nuclear antigen
(PCNA). The primary function of ring-shaped sliding
clamps is to tether Pols to the template and increase their
processivity (13–15). Sliding clamps also function in other
genome replication steps, as well as in recombination, repair and cell cycle regulation by interacting with diverse
proteins involved in these processes (16,17). For example,
sliding clamps associate with the mismatch repair (MMR)
proteins bacterial MutS and eukaryotic MutS␣ (13,18–21).
Most of the sliding clamps-binding proteins interact with a
hydrophobic cleft on the surface of the ring via a conserved
clamp binding motif (CBM) (17,22). In addition, given
the homopolymeric structure of sliding clamps, dimeric ␤
clamp and trimeric PCNA contain two or three binding
sites, respectively (16,17). Thus, partner proteins can simultaneously interact with or compete for binding the processivity factors. Sliding clamps also play important roles in
regulating the action and activity of their partners on DNA.
The lesion bypass and mutagenic activities of TLS Pol II,
Pol IV and Pol V have been shown to be completely dependent upon the association with ␤ clamp, which targets these
Pols to replication sites (23,24).
How Pol IV gains access to ␤ clamp has been extensively
studied in Pol III HE replication assays, since it has been
proposed that Pol IV activities (TLS and induction of mutagenesis by its overproduction) take place at the replication
fork. In TLS reactions reconstituted on DNA templates
containing a N2 -dG adduct, processive DNA synthesis by
Pol III HE is blocked at the lesion site (25,26). Addition
of Pol IV alleviates the block and results in the switching
from Pol III HE to Pol IV, which performs the bypass reaction. Pol IV is also able to gain control of ␤ clamp and the
primed-DNA when Pol III HE is stalled due to nucleotide
omission and, much less efficiently, during active Pol III HE
replication of undamaged DNA (26–29). After DNA synthesis by Pol IV, Pol III HE rapidly regains control of the
primed-DNA from Pol IV and resumes processive replication. What are the factors controlling the switches between
Pol III HE and Pol IV? Structural changes in Pol III HE
and/or ␤ clamp, induced by the replication stall or a DNA
lesion, may destabilize the association of Pol III HE with
␤ clamp and the primer terminus, and may unmask surfaces of interaction with Pol IV. It has been demonstrated
that the Pol exchange involves a specific interaction of Pol
IV with a secondary surface of ␤ clamp in addition to the
CBM-cleft contact (27,30). A direct interaction of Pol IV
with Pol III HE is also likely to contribute to Pol switching
(31,32). Thus, a combination of these interactions has been
suggested to be necessary to recruit Pol IV to the stalled
replisome (32). Interactions between ␤ clamp and the DNA
template that it encircles may represent an additional level
of regulation of the switching. ␤ clamp–DNA interactions
involve the cleft and two additional surfaces of the ring
(33), one of which (residues H148-R152) is also required for
binding to Pol IV (34). It is expected that ␤ clamp senses the
damaged DNA or the replication stall and alters its interaction with the DNA template to enable contact with Pol
IV, thereby allowing access of Pol IV to the primer terminus. Notably, other proteins may impose a regulatory control over Pol IV binding to ␤ clamp.
The association between the MMR protein MutS and
␤ clamp has been extensively studied in bacteria and eukaryotes. However, a limited contribution of this interaction
in the currently recognized functions of the MMR system,
namely the correction of replication errors and inhibition of
homologous recombination, has been shown, which raises
questions about their functional significance in these organisms (19–21,35). In this work, we present for the first time
evidence that MutS from Pseudomonas aeruginosa inhibits
Pol IV–␤ clamp interaction by competing for the hydrophobic cleft of the ring in vitro. Moreover, MutS–␤ clamp interaction is critical for controlling of the Pol IV mutagenic
activity in exponentially growing P. aeruginosa cells. Thus,
our findings reveal a key role of MutS in the regulation of
the error-prone Pol IV access to replication sites, which corresponds to a novel mechanism for maintaining replication
fidelity by MutS.
MATERIALS AND METHODS
Bacterial strains
The experiments were carried out using a P. aeruginosa
PAO1 strain and its isogenic mutS␤ derivative (19). An
SOS non-inducible PAO1 mutant strain, constructed by inserting the non-cleavable lexAG86V allele in the chromosome of a lexA null mutant, was kindly provided by Dr
Pradeep Singh from the University of Washington School
of Medicine (36).
Construction of plasmids and chromosomal PAO1 mutant
strains, expression and purification of the recombinant proteins, and western blot assays
These experimental procedures are detailed in the Supplementary Data.
Native polyacrylamide gel electrophoresis assays
Native polyacrylamide gel electrophoresis (PAGE) assays
were carried out by incubating affinity-purified proteins or
the MQSDLFA peptide (Peptide 2.0 Inc) in the presence or
absense of DNA oligonucleotides in 20 mM Tris–HCl, pH
7.4, amended with 5 mM MgCl2 , 1 mM dithiothreitol, 25
mM NaCl and 100 ␮g/ml BSA for 25 min at 30◦ C. The samples were applied to native polyacrylamide gels prepared in
Tris-borate buffer and run at 4◦ C. The gels were Coomassie
stained and the protein bands were quantified using the GelPro analyzer software. Additional details are described in
the Supplementary Data.
7702 Nucleic Acids Research, 2016, Vol. 44, No. 16
Surface plasmon resonance assays
Surface plasmon resonance (SPR) analysis was performed
using a BIAcore T100 instrument (GE Healthcare). Proteins were immobilized on a CM5 chip (GE Healthcare)
surface by amine coupling according to the manufacturer’s
instructions. Soluble proteins were diluted in the Tris 20
mM, pH 7.4, NaCl 25 mM running buffer and injected over
chip surfaces at a flow rate of 30 ␮l min−1 for 45 or 90 s at
37◦ C. Additional details are described in the Supplementary Data.
Dot far-western blotting assays
These assays were performed as previously described (19)
with minor modifications as detailed in the Supplementary
Data.
Estimation of spontaneous mutation rates and analysis of
nfxB mutation spectra
Mutation rates were determined by the modified Luria–
Delbruck fluctuation test (37). nfxB sequence was analyzed
from ciprofloxacin resistant colonies (38). Additional details are described in the Supplementary Data.
Transcriptional analysis of the lexA promoter
To evaluate SOS-induction, a transcriptional fusion of the
lexA promoter to the luxCDABE reporter operon was inserted into the P. aeruginosa chromosome. Luminescence
was measured in exponentially growing cells as follow: cultures were diluted to an OD 600 nm of 0.10 and seeded in 96well plates (100 ␮l). The 96-well plate was then imaged by
integrating luminescence signal for 20 s in a NightOWL LB
983 instrument, and the number of photons emitted per well
was quantified using the Berthold WinLigth 32 Software. To
evaluate luminescence of colony forming units, plates were
imaged using a 1 min exposure time.
N-ethyl-N-nitrosourea sensitivity
Cells were inoculated in LB medium and grown to exponential phase. Cells freshly transformed with the derivatives of
p5BAD were cultured in LB medium containing 5 ␮g/ml
gentamicin. Aliquots from successive dilutions were plated
onto LB agar supplemented or not with 0.6 mM N-ethylN-nitrosourea (ENU), followed by overnight incubation at
37◦ C. Colony forming units were counted, and survival was
determined relative to the LB control, which was set equal
to 100%. Transcription from the lexA promoter was assayed
by monitoring the luminescence of colonies from cells containing the lexA::luxCDABE reporter.
RESULTS
Pol IV binds to ␤ clamp, albeit with lower affinity than MutS
Pseudomonas aeruginosa MutS (855 amino acids) interacts
with ␤ clamp via the 816 QSDLF820 binding site located at its
C terminal domain (19). Change of this motif to ASDAA,
obtaining a mutant version denominated MutS␤ , abolishes
the association with the replicative factor. Interaction between P. aeruginosa Pol IV and ␤ clamp has not previously
been examined.
Association of Pol IV or MutS with ␤ clamp was initially
evaluated by native PAGE (Figure 1A and B). Increasing
concentrations of Pol IV or MutS were incubated with a
fixed amount of ␤ clamp. The intensity of the band corresponding to free ␤ clamp decreased with increasing Pol IV
or MutS concentrations (Supplementary Figure S1A and
B). The plot of the ␤ clamp bound fraction as a function
of free Pol IV concentrations fitted to a rectangular hyperbola indicating a single binding site (n = 1.09 ± 0.06) on
dimeric ␤ clamp (Figure 1A). The sigmoidal-shaped Klotz
plot and the linear-shaped Scatchard plot of the same data
also suggested a single binding site (data not shown). The
KD value for the Pol IV–␤ clamp interaction was 6.22 ±
0.51 ␮M. The MutS–␤ clamp binding data fitted a sigmoidal curve (Figure 1B). Analysis of this binding data by
the Hill equation revealed a positive cooperative interaction, which was confirmed by the concave-downward curve
of the Scatchard plot (data not shown). The Hill coefficient
was 4.53 ± 1.18 indicating that four MutS molecules bind
per ␤ clamp molecule, correlating with the fact that MutS
can form tetramers (39). The estimated KD by Hill plot analysis was 1.66 ± 0.09 ␮M.
The estimated strengths of Pol IV–␤ clamp and MutS–
␤ clamp interactions in the native PAGE assays were also
observed by SPR (Figure 1C and D). Pol IV specifically
bound to ␤ clamp immobilized to a CM5 chip with a KD =
8.97 ␮M (Figure 1C). MutS was also able to associate with
immobilized ␤ clamp (Supplementary Figure S2), however
it was not possible to accurately determine a KD value for
the MutS–␤ clamp interaction. To further characterize the
MutS–␤ clamp affinity, ␤ clamp was captured using an antibody conjugated to a CM5 chip and then solutions of MutS
were made to flow over it. MutS was bound to ␤ clamp 9fold more strongly than Pol IV did, with KD = 0.99 ␮M
(Figure 1D). We also examined these interactions by dot
far-western blotting analysis (Supplementary Figure S3A).
Binding of soluble ␤ clamp to MutS immobilized on nitrocellulose was detected at all tested amounts, and sharply increased between 0.05 and 1.45 pmol of MutS. In contrast,
interaction between soluble ␤ clamp and immobilized Pol
IV was not observed until Pol IV concentration reached 0.73
pmol. Taken together, our findings clearly indicate that the
binding affinity of MutS for ␤ clamp is higher than that of
Pol IV for ␤ clamp.
P. aeruginosa Pol IV (349 amino acids) contains the
345
QLRLF349 hydrophobic cluster at its extreme C-terminus
that resembles the consensus ␤ clamp-binding motif
QL[S/D]LF identified by bioinformatics analysis of eubacterial ␤ clamp-binding proteins (22). To establish if Pol IV
uses this conserved motif to interact with ␤ clamp, a Pol
IV mutant version was constructed by deleting the five QLRLF amino acids, herein referred as Pol IV-5, and tested
for binding to ␤ clamp. Pol IV-5, as well as MutS␤ , failed
to associate with ␤ clamp in the native PAGE (Figure 1A
and B; Supplementary Figure S1C and D) and the dot farwestern blotting assay (Supplementary Figure S3A). Thus,
these results indicate that the QLRLF conserved binding
motif represents the main site for Pol IV binding to ␤ clamp.
Nucleic Acids Research, 2016, Vol. 44, No. 16 7703
Figure 1. Analysis of Pol IV–␤ clamp and MutS–␤ clamp interactions. Saturation binding curves for Pol IV (A) and MutS (B). Pol IV (1–45 ␮M), Pol
IV-5 (4–32 ␮M), MutS (1–14 ␮M) and MutS␤ (2–14 ␮M) were incubated with a fixed concentration of ␤ clamp (2 ␮M), and reaction products were
analyzed in a native polyacrylamide gel. Saturation binding curves were obtained by plotting the fraction of ␤ clamp bound versus free concentrations of
these proteins. A representative experiment is shown (n = 3). (C) Pol IV–␤ clamp interaction. ␤ clamp was conjugated to a CM5 chip, followed by injection
of Pol IV (0.5–4.0 ␮M) in SPR-based assays. Kinetic constants for the Pol IV–␤ clamp interaction were kon = (5.80 ± 0.10) 102 M−1 s−1 and koff = (5.20 ±
0.20) 10−3 s−1 . (D) MutS–␤ clamp interaction. ␤ clamp was antibody-captured, followed by injection of MutS (35–280 nM) in SPR-based assays. Kinetics
constants for MutS binding to ␤ clamp were kon = (1.12 ± 0.08) 105 M−1 s−1 and koff = (1.11 ± 0.04) 10−1 s−1 . Sensograms of MutS displays solvent
correction. Representative reference-subtracted curves are shown (n = 3).
Finally, as a control, interaction between MutS and Pol
IV was examined by SPR (Supplementary Figure S4A and
B) and dot far-western blotting (Supplementary Figure
S4C–E). No binding was detected between Pol IV and MutS
in these conditions.
MutS inhibits Pol IV interaction with ␤ clamp by competing
for the protein-binding cleft of the ring
We asked if MutS affects the interaction of Pol IV with ␤
clamp. To examine this issue, Pol IV was immobilized to
the CM5 chip and then ␤ clamp alone or pre-incubated
with increasing MutS concentrations (30–250 nM) was injected. ␤ clamp interacted with Pol IV, but this association was partially inhibited at 30 nM MutS and abolished by higher MutS concentrations (Figure 2A). Conversely, ␤ clamp binding to Pol IV was not prevented by
pre-incubation with 250 nM MutS␤ , which did not interact with ␤ clamp in the SPR assays (Supplementary Figure S5). We also examined the formation of the Pol IV–␤
clamp complex by native PAGE when ␤ clamp, Pol IV and
MutS were simultaneously mixed (Figure 2B and Supplementary Figure S1E). MutS, but not MutS␤ , hampered the
Pol IV–␤ clamp interaction. At 1 ␮M MutS, the fraction
of ␤ clamp bound to Pol IV was ∼0.45 relative to the control in the absence of MutS, whereas the formation of the
Pol IV–␤ clamp complex was completely abolished at 3 ␮M
MutS. Similar results were obtained in the dot far-western
blotting assay (Supplementary Figure S3B). Interaction of
soluble ␤ clamp with immobilized Pol IV decreased as increasing amounts of MutS were added into solution. This
effect was not detected with the mutant MutS␤ version. We
next asked if MutS is able to disrupt the association of Pol
IV with ␤ clamp. To further evaluate this question, the Pol
IV–␤ clamp complex was formed and then MutS was added
(Figure 2C and Supplementary Figure S1F). The fraction
of ␤ clamp bound to Pol IV was reduced following MutS
addition as revealed by native PAGE. We only detected a
small fraction (∼0.18) of ␤ clamp bound to Pol IV at 3 ␮M
MutS. Addition of the mutant MutS␤ version did not interrupt the Pol IV–␤ clamp association. Taken together, these
results demonstrate that MutS inhibits the formation of the
Pol IV–␤ clamp complex and, is also able to displace Pol IV
from the ring.
Finally, we examined whether MutS and Pol IV bind to ␤
clamp at the same site by analyzing if a 7-mer peptide spanning the conserved ␤ clamp binding motif located at the C
terminus of MutS (815 MQSDLFA821 , underlined residues
represent the CBM) impairs the Pol IV–␤ clamp interaction.
Addition of this MutS-derived peptide inhibited binding of
Pol IV to ␤ clamp in the native PAGE assay. The result, in
Figure 3, shows an increase in the intensity of the band corresponding to the ␤ clamp–peptide complex as the peptide
7704 Nucleic Acids Research, 2016, Vol. 44, No. 16
Figure 3. A peptide spanning the ␤ clamp conserved motif of MutS inhibits the interaction of Pol IV with ␤ clamp. Pol IV (20 ␮M) and MutS
(6 ␮M) were individually incubated with ␤ clamp (2 ␮M) in the presence
of increasing amounts of the MQSDLFA peptide. Concentrations of the
peptide were 10-, 50- and 100-fold higher than Pol IV and MutS concentrations. Reaction products were analyzed by a native PAGE. The upper
panel shows the band corresponding to the ␤ clamp–peptide complex. The
intensity of the ␤ clamp–peptide complex bands was measured, and the
fraction of ␤ clamp bound to peptide was calculated taken as 1 the intensity of the band obtained from a reaction wherein ␤ clamp was mixed with
the peptide (lower panel). Data represent mean ± standard deviation (n =
3).
DNA structures modulate the ability of MutS to inhibit Pol
IV–␤ clamp interaction
Figure 2. Effect of MutS on the Pol IV–␤ clamp interaction. (A) Interaction of Pol IV with ␤ clamp complexed to MutS. Pol IV was conjugated
to a CM5 chip, followed by injection of ␤ clamp (0.25 ␮M) alone or preincubated with MutS (30, 60, 125, 190 and 250 nM) in SPR-based assays.
Representative reference-subtracted curves are shown (n = 3). (B) MutS
inhibited Pol IV–␤ clamp interaction. Pol IV (20.0 ␮M), ␤ clamp (2.0 ␮M)
and increasing concentrations of MutS or MutS␤ (1.0, 2.0 and 3.0 ␮M)
were simultaneously incubated. (C) MutS displaced Pol IV from ␤ clamp.
Pol IV (20.0 ␮M) and ␤ clamp (2.0 ␮M) were pre-incubated and then MutS
or MutS␤ (1.0, 2.0 and 3.0 ␮M) were added. Reaction products were analyzed by native polyacrylamide gel electrophoresis (PAGE). Band intensities of the Pol IV–␤ clamp complex were quantified and used to calculate
the fraction of ␤ clamp bound to Pol IV taken as 1 the intensity of bands in
the control condition corresponding to incubation of Pol IV with ␤ clamp
alone. Data represent mean ± standard deviation (n = 3).
was titrated into the reaction (Supplementary Figure S1G).
As expected, the peptide also impaired the association of
MutS with ␤ clamp (Figure 3 and Supplementary Figure
S1H). These data further support the conclusion that MutS
and Pol IV share a common site on ␤ clamp, namely the
hydrophobic cleft between domains II and III of the ␤ ring
(40).
Biochemical studies showed that MutS strongly interacts
with heteroduplex DNA and weakly binds homoduplex
DNA (19,41). ␤ clamp binds to primed-DNA and homoduplex DNA (33), and Pol IV shows a low affinity for primedDNA (15). In addition, it has been reported that the presence of DNA substrates affects the affinity between ␤ clamp
and its interaction partners (42). Therefore, we examined
the effect of different DNA substrates on the affinity of Pol
IV–␤ clamp and MutS–␤ clamp interactions (Figure 4A).
To further investigate this, we mixed these proteins in the
presence of single-stranded DNA, homoduplex DNA, GT
heteroduplex DNA, primed-DNA or GT primed-DNA.
Then, binding was analyzed using native PAGE. The results
show that neither DNA substrates had a significant effect on
the strength of the Pol IV–␤ clamp interaction. Strikingly,
the MutS–␤ clamp interaction was enhanced in the presence
of the GT primed-DNA, as shown by the 3.5-fold increase
in the fraction of bound ␤ clamp compared to the control
reaction without DNA. This effect was specific for the GT
primed-DNA since the MutS–␤ clamp interaction did not
change when the other DNA substrates were added to the
reaction. As a control, and to analyze if the GT primedDNA favored the MutS–␤ clamp association by bringing
together both proteins onto this DNA structure, we tested
the binding between the MutS␤ mutant and ␤ clamp in the
presence of this DNA substrate. MutS␤ failed to associate
with ␤ clamp under this condition (Supplementary Figure
Nucleic Acids Research, 2016, Vol. 44, No. 16 7705
fractions were bound to Pol IV (∼0.56 and ∼0.60, respectively). The Pol IV–␤ clamp association was not inhibited by
MutS in the presence of the single-stranded DNA (∼1.04),
demonstrating that the capability of MutS for preventing
the formation of the Pol IV–␤ clamp complex is suppressed
by this DNA structure. Conversely, the primed-DNA and
the GT primed-DNA improved the ability of MutS to inhibit the Pol IV–␤ clamp interaction as the fraction of ␤
clamp bound to Pol IV was reduced following addition of
these DNA substrates (∼0.30 and ∼0.45, respectively). The
enhanced capability of MutS for impairing the Pol IV–␤
clamp interaction in the presence of primed-DNAs was also
observed by SPR (Supplementary Figure S7). Reactions of
␤ clamp alone or pre-incubated with the primed-DNA substrates were mixed with MutS (30 or 60 nM) and then injected over Pol IV immobilized to a CM5 chip. ␤ clamp
bound to immobilized Pol IV. This interaction was prevented when the processivity factor was mixed with 60 nM
MutS, but it was not inhibited at 30 nM MutS. Addition
of the primed-DNA or the GT primed-DNA substrates to
reactions containing 30 nM MutS abolished ␤ clamp binding to Pol IV. Taken together, these results show that specific DNA structures regulate the effectiveness of MutS as
a competitor for the Pol IV–␤ clamp interaction.
Pol IV strongly contributes to spontaneous mutagenesis in P.
aeruginosa cells expressing MutS␤
Figure 4. Effect of DNA substrates. (A) Pol IV–␤ clamp and MutS–␤
clamp interactions. Pol IV (2 ␮M) and MutS (1 ␮M) were individually
mixed with ␤ clamp (2 ␮M) in the presence of the DNA substrates (15
␮M). Reaction mixtures were analyzed by native PAGE. The intensity of
the bands corresponding to free ␤ clamp (upper panel) was quantified and
used to calculate the fraction of ␤ clamp bound (lower panel). (B) MutS
inhibition of the Pol IV–␤ clamp interaction. Spots of Pol IV (3 pmol) on
nitrocellulose membranes were incubated with soluble ␤ clamp (0.25 ␮M,
N-terminally His6 -tagged) mixed with MutS (0.50 ␮M) and the DNA substrates (15 ␮M). ␤ clamp binding was detected using a mouse anti-His6
monoclonal antibody. The intensity of the spots was quantified and used
to calculate the fraction of ␤ clamp bound to Pol IV taken as 1 the intensity
of spots in the control condition corresponding to soluble ␤ clamp alone.
The DNA substrates used were a single-stranded DNA (ssDNA), homoduplex DNA (dsDNA), GT heteroduplex DNA (GT dsDNA), primed-DNA
or GT primed-DNA. A control reaction without DNA was also included.
Data represent mean ± standard deviation (n = 3).
S6), discarding this possibility. These data indicate that a
primed-DNA containing a GT mispair specifically modulates the affinity between MutS and ␤ clamp.
We next examined whether the ability of MutS to inhibit
Pol IV binding to ␤ clamp is regulated by these DNA structures. This issue was first evaluated by dot far-western blotting (Figure 4B). The fraction of soluble ␤ clamp bound
to immobilized Pol IV was reduced when MutS was added
into solution (∼0.59 relative to the control in the absence of
MutS). The homoduplex DNA and GT heteroduplex DNA
had no effect on this MutS ability since similar ␤ clamp
Pol IV overexpression. Pol IV overexpression was shown
to result in a weak mutagenesis of chromosomal genes in E.
coli (9–10,43), Pseudomonas putida (11) and P. aeruginosa
(5). This mutator phenotype is dependent upon the ability
of Pol IV to interact with ␤ clamp since this factor targets
Pol IV to replication sites, resulting in the low fidelity replication of undamaged DNA (24).
To ascertain whether MutS is able to control interaction between Pol IV and ␤ clamp, and consequently the
access of this Pol to replication sites, we examined the effect of Pol IV overexpression on mutagenesis in exponentially growing P. aeruginosa PAO1 cells from a mutS␤ mutant strain, in which the chromosomal mutS allele was replaced by mutS␤ that encodes MutS␤ (19), compared with
the parental (WT) strain. We tested mutations in different target genes on the P. aeruginosa chromosome by measuring mutation rates to resistance to ciprofloxacin (Cipr ;
nfxB, gyrA and parC are mutated), rifampicin (Rifr ; rpoB
is mutated) and amikacin (Amkr ; mexZ is mainly mutated)
(38,44–46). Overproduction of this Pol was carried out by
cloning the dinB gene encoding Pol IV into an arabinose
inducible plasmid p5BAD (see Supplementary Data). Expression of the ectopic dinB gene was detected in mutS␤ and
WT cells harboring p5BAD-dinB after induction with arabinose (Supplementary Figure S8A and B, lines 2–7). This
protein was not detected in arabinose-induced cells carrying the empty vector p5BAD (Supplementary Figure S8A
and B, line 1). A linear relationship between arabinose concentrations (0.005–0.050%) and Pol IV levels was observed
(Supplementary Figure S8E). In addition, similar curves
were obtained with the mutS␤ and WT strains (Supplementary Figure S8E), indicating that there are not significant
differences in the induction of Pol IV in both strains.
7706 Nucleic Acids Research, 2016, Vol. 44, No. 16
Pol IV overexpression from plasmid p5BAD-dinB increased mutation rates in the WT and mutS␤ strains relative
to control cells harboring the empty vector p5BAD (Figure
5A and B; Supplementary Figure S9). It should be noted
that equal mutation rates were observed for cells with or
without p5BAD (Figure 5B and Supplementary Figure S9C
and D). The WT strain displayed 4-, 3- and 2-fold higher
mutation rates to Cipr , Rifr and Amkr , respectively, independently of Pol IV expression levels (Figure 5A; Supplementary Figure S9A and B). Thus, although addition of
0.050% arabinose resulted in a 10-fold higher Pol IV expression than that obtained with 0.005% arabinose (Supplementary Figure S8E), there were no significant differences in
mutation rates at all arabinose concentrations tested. In the
mutS␤ strain, mutagenesis was enhanced as Pol IV expression levels increased reaching 22-, 15- and 17-fold higher
mutation rates to Cipr , Rifr and Amkr after induction with
0.050% arabinose, respectively (Figure 5A; Supplementary
Figure S9A and B). Then, we measured mutation rates in
the WT and mutS␤ strains containing p5BAD derivatives
encoding Pol IV-5 or the catalytic-deficient Pol IV-D8A
mutant. Pol IV-D8A had a substitution of the conserved aspartic acid residue to alanine at position 8 in the polymerase
catalytic site I, which severely impairs the polymerase activity (5). Introduction of p5BAD plasmids carrying dinB
alleles coding for these mutant versions failed to enhance
the mutation rates after induction with 0.050% arabinose in
both strains (Figure 5B; Supplementary Figure S9C and D).
Quantification by western blotting revealed that expression
levels of both Pol IV mutants were not comparable to that of
the wild-type protein at 0.050% arabinose (Supplementary
Figure S8A and B, lines 8 and 9). Pol IV-5 was present at 4and 6-fold higher expression levels and Pol IV-D8A showed
a 4- and 10-fold decrease in the WT and mutS␤ strains, respectively. It should be noted that these Pol IV-D8A levels
were similar to that obtained of wild-type Pol IV at lower
arabinose concentrations (0.005 and 0.010%) wherein mutagenesis was increased.
These findings demonstrate that Pol IV ectopically expressed from plasmid is able to introduce mutations on the
P. aeruginosa chromosome, and this Pol IV-induced mutagenesis is considerably higher in the mutS␤ strain to that
observed in the WT strain. As previously observed (5,24),
both the Pol activity and the interaction with the processivity factor are required to enhance mutagenesis by Pol IV.
Mutation spectra. We next investigated the Pol IV mutational specificity in the mutS␤ background compared with
the WT background by characterizing the mutation spectra of the reporter gene nfxB, which scores a broad range
of mutations and allows the analysis of strand bias and preferred sequence contexts of mutation (38). Sequencing of
the entire open reading frame and promoter region of nfxB
from Cipr clones showed that the six base substitutions, 1bp deletions and insertions and >1-pb deletions were detected within this gene in all PAO1 strains (Supplementary
Table S1). When the nfxB mutation spectra from the mutS␤
and WT strains were compared, there was a significant 2fold increase in the rate of the AT > CG base substitution
in the mutS␤ strain (Figure 5C and Supplementary Table
S1). To examine if the increase of this transversion is due
to the Pol IV activity, we constructed Pol IV-deficient cells
by deleting dinB from the mutS␤ and WT strains. We did
not detect any difference in mutation rates to Cipr among
the WT, mutS␤ , dinB and mutS␤ dinB strains (Supplementary Table S1). However, deletion of dinB significantly decreased the rate of AT > CG (2-fold), CG > AT (2-fold)
and AT > GC (3-fold) in the mutS␤ background (mutS␤
versus mutS␤ dinB strains) (Figure 5C and Supplementary
Table S1). Notably, Pol IV-deficiency did not decrease the
rate of any mutations in the WT background (WT versus
dinB strains) (Figure 5C and Supplementary Table S1). Pol
IV overexpression mainly promoted AT > GC, AT > CG,
CG > GC and >1-bp deletions in the mutS␤ and WT strains
compared with control empty cells (Supplementary Table
S1). The rate of these mutations were 13-, 9-, 3- and 6-fold
higher in the mutS␤ strain relative to that observed in the
WT strain (Supplementary Table S1). Finally, we corroborated that these mutations resulted from the Pol IV activity
since overexpression of the polymerase activity-deficient Pol
IV-D8A mutant did not change the nfxB mutation spectra
in the WT and mutS␤ strains (data not shown).
These data indicate that mutational specificity of P.
aeruginosa Pol IV is similar to that of E. coli Pol IV. Thus,
Pol IV overexpression in both bacteria preferentially promotes base substitutions toward GC pairs (43). In addition, and accordingly to the previous data (5,10), Pol IV
does not contribute to spontaneous mutagenesis in exponentially growing cells since inactivation of dinB does not
change the mutation spectra in the WT background. On the
other hand, a proportion of spontaneous mutations are generated by this Pol in the mutS␤ strain, and overproduction
of Pol IV enhances to a greater extent some base substitutions and deletions in this strain.
Strand bias. It was previously observed that overproduction of Pol IV enhances preferentially mutations during lagging strand synthesis in E. coli (9). To explore if there is a
differential mutator effect of Pol IV in the two replicating
strands of the P. aeruginosa chromosome, we analyzed each
nfxB base substitution spectrum for strand bias using the
iMARS software (47) (Supplementary Table S2). The mutation spectra from the WT strain showed no strand bias.
Overexpression of Pol IV from the p5BAD-dinB plasmid
caused a strand bias whereas the deficiency of this Pol (dinB
strain) had no effect in the WT background. A significant
strand bias was evident in the mutation spectra from the
mutS␤ strain. Deletion of dinB (mutS␤ dinB strain) eliminated this strand bias. As expected, mutS␤ cells harboring p5BAD-dinB retained the strand bias observed in the
mutS␤ strain. Thus, a preferential mutagenesis on one of
the strands of the replication fork was evident in exponentially growing cells of the mutS␤ strain, but not in the WT
strain, which was dependent on Pol IV.
Sequence context specificity. We found that the GG and
GC dinucleotides were most frequently mutated in the nfxB
base substitution spectra of the mutS␤ and WT strains harboring p5BAD-dinB using iMARS (data not shown). Both
dinucleotides were almost exclusively present in sites undergoing CG > GC transversions, which corresponds to one
of the most promoted mutations by Pol IV overexpression
Nucleic Acids Research, 2016, Vol. 44, No. 16 7707
Figure 5. Contribution of Pol IV to spontaneous mutagenesis. (A and B) Mutation rates to Cipr per replication and 95% confidence limits were calculated
as described in ‘Materials and Methods’ section. (A) WT and mutS␤ strains carrying p5BAD-dinB after induction with different arabinose concentrations
(0.005–0.050%). (B) WT and mutS␤ strains without plasmid or carrying p5BAD, p5BAD-dinB, p5BAD-dinBD8A or p5BAD-dinB5 after induction with
0.050% arabinose. (C) Rates of each mutation type detected in nfxB from Cipr clones were compared between the WT, dinB, mutS␤ and mutS␤ dinB strains.
Error bars represent the upper and lower 95% confidence intervals. No overlap of 95% confidence intervals indicates statistically significant differences
(marked with asterisks in panel C).
(Supplementary Tables S3 and 4). Visual inspection of sequences surrounding the mutated nucleotide revealed that
this mutation mainly occurred in the GGC trinucleotide
(which included the GG and GC dinucleotides; the mutated
nucleotide is indicated in bold). In addition, a high proportion of the AT > CG transversions occurred in the GAC
trinucleotide. Thus, the GXC sequence corresponded to a
preferred context for Pol IV within nfxB. This trinucleotide
was detected in 58 and 80% of the base substitutions for
the mutS␤ and WT strains containing p5BAD-dinB, respectively (Supplementary Tables S3 and 4). In the WT strain,
only 3% of base substitutions occurred within GXC sequences whereas a major proportion (14%) of these mutations was observed in this sequence context in the mutS␤
strain (Supplementary Tables S5 and 6). In contrast, base
substitutions were not present at sites involving the GXC
sequence in the dinB strain (Supplementary Table S7). This
trinucleotide was detected in 4% of the base substitutions in
the mutS␤ dinB strain (Supplementary Table S8).
The sequence specificity of base substitutions induced by
P. aeruginosa Pol IV resembles to that observed for the E.
coli enzyme, which preferred the 5 -GX-3 context where X
represents the mutated base within the cII and rpoB genes
(10,43). Moreover, our data show that the GXC sequence
context is more frequently represented in the mutation spectrum of the mutS␤ strain when compared to that observed
in the WT strain, and this depends on the presence of Pol
IV.
Pol IV-induced mutagenesis is not controlled by MutS under
SOS conditions
Pol IV expression is induced as part of the LexA-dependent
SOS regulon in E. coli and related bacteria as a response
to environmental stress (48). Inactivation of the LexA repressor by a RecA-mediated autocleavage after DNA damage leads to the transcriptional de-repression of the SOSregulated genes including dinB and lexA. In P. aeruginosa,
dinB transcription levels are increased upon disruption of
lexA, confirming that LexA acts to repress dinB transcription (5).
We asked if MutS is able to control Pol IV-dependent mutagenesis under SOS-induced conditions. To examine this
issue, LexA-deficient derivatives of the WT, mutS␤ , dinB
and mutS␤ dinB strains were constructed by replacing the
chromosomal lexA gene with an inactive allele (see Supplementary Data). In addition, and in order to measure
the relative induction of the SOS gene network, a reporter
construction containing the putative promoter region of
lexA cloned upstream of the luxCDABE operon was inserted into the chromosome of strains harboring an active
(lexA+) or inactive (lexA-) allele. An ∼3-fold higher luminescence signal was detected in exponentially growing cultures of the lexA- strains relative to that observed in the
lexA+ strains (Figure 6A), confirming that LexA deficiency
induces the SOS response. Then, we measured mutation
rates to Cipr to test mutation levels in cells expressing constitutively the SOS response. lexA inactivation significantly
increased mutagenesis in the WT and mutS␤ backgrounds
(Figure 6B). lexA and mutS␤ lexA strains showed similar
7708 Nucleic Acids Research, 2016, Vol. 44, No. 16
Figure 6. Pol IV-induced mutagenesis under SOS conditions. (A) Transcriptional analysis of the lexA promoter. Transcription from the lexA promoter was
assayed by measuring the luminescence in exponentially growing cultures of cells harboring a transcriptional fusion of the lexA promoter to the luxCDABE
reporter operon inserted into the chromosome. Data represent mean ± standard deviation (n = 3). (B) Mutation rates to Cipr per replication and 95%
confidence limits were calculated as described in ‘Materials and Methods’ section. Error bars represent the upper and lower 95% confidence intervals.
No overlap of 95% confidence intervals indicates statistically significant differences. The strains used were WT, mutS␤ , dinB and mutS␤ dinB harboring an
active (lexA+) or inactive (lexA−) allele in the chromosome. These strains also contained the reporter fusion inserted into the chromosome. It should be
noted that equal mutation rates were observed for the lexA+ strains with or without the reporter fusion.
mutation rates, which were 11- and 16-fold higher compared
to WT and mutS␤ , respectively. Deletion of dinB considerably decreased mutagenesis under SOS conditions (Figure
6B). dinB lexA and mutS␤ dinB lexA strains displayed 3.5and 3.4-fold reduced mutation levels relative to lexA and
mutS␤ lexA, and they exhibited 3.2- and 3.5-fold higher mutation rates compared with dinB and mutS␤ dinB. These results indicate a key role for Pol IV in SOS-induced mutagenesis, as it was observed in E. coli (49). Our data also clearly
demonstrate that MutS does not limit Pol IV-dependent
mutagenesis under SOS conditions since mutation levels in
the WT and mutS␤ backgrounds are similar.
The capability of Pol IV for catalyzing TLS is not affected in
the mutS␤ background
Pol IV is involved in accurate bypass of DNA lesions induced by alkylating agents and thus, confers resistance to
the killing effect of exogenous alkylating compounds such
as ENU (6). To analyze the capacity of Pol IV to participate in TLS events in the WT and mutS␤ backgrounds,
we tested the survival of exponentially growing cells after
treatment with ENU (Figure 7). Exposure to ENU reduced
cell viability of the WT and mutS␤ strains to the same extent. Cell viability was reduced to 15 and 12% of control
untreated WT and mutS␤ cells after exposure to ENU, respectively. As expected, Pol IV-deficient cells from the WT
and mutS␤ strains were ∼7-fold more sensitive. Pol IV overexpression increases cell survival in both strains. ENU sensitivity of the WT strain bearing p5BAD-dinB was similar
to that of the mutS␤ strain containing the Pol IV-expressing
plasmid. About 42 and 54% of WT and mutS␤ cells harboring p5BAD-dinB survived after the treatment with ENU,
respectively. Conversely, ENU sensitivity of the WT and
mutS␤ strains bearing p5BAD was comparable to that of
empty cells. Exposure to this alkylating agent did not induce
the SOS response as it was tested using the luminescent reporter of the lexA promoter transcription (Supplementary
Figure S10). The WT, mutS␤ , dinB and mutS␤ dinB strains
showed similar luminescence levels to a SOS-deficient PAO1
strain expressing the uncleavable LexAG86V version (36) in
plates containing ENU. These findings suggest that disrup-
Figure 7. Analysis of sensitivity to ENU-induced DNA lesions. Sensitivity to ENU was measured in cells containing the chromosomal dinB allele
(dinB+, WT and mutS␤ strains) or carrying a deletion of dinB (dinB-, dinB
and mutS␤ dinB strains), and WT and mutS␤ cells containing the empty
p5BAD or p5BAD-dinB. Data represent mean ± standard deviation (n =
3).
tion of the MutS–␤ clamp interaction does not appear to
affect the ability of Pol IV to bypass ENU-induced lesions.
This uncontrolled action of Pol IV by MutS may be owing to that Pol IV replication of ENU lesions is error-free,
and not to induction of the SOS response by the alkylating
agent.
MutS and Pol IV expression levels in exponentially growing
cells
MutS and MutS␤ levels were measured by western blot
assays in WT and mutS␤ cells, respectively, carrying a Cterminal fusion of the chromosomal encoded MutS or
MutS␤ to a Strep II tag (Supplementary Figure S11). Cellular levels of MutS were indistinguishable from that of
MutS␤ in exponentially growing cells expressing Pol IV
basal levels, as well as when Pol IV and Pol IV-5 were overexpressed from p5BAD after induction with 0.050% arabinose. These assays confirm that MutS and MutS␤ expression are similar and thus, the major impact of Pol IV on mutagenesis in the mutS␤ background is not due to differences
between MutS and MutS␤ levels. Likewise, Pol IV expres-
Nucleic Acids Research, 2016, Vol. 44, No. 16 7709
sion levels were tested in WT and mutS␤ cells carrying an
N-terminal fusion of the chromosomal encoded Pol IV to a
His6 tag. In both strains, Pol IV was not detected by western
blotting using polyclonal or monoclonal antibodies raised
against the His6 tag (data not shown). This is in agreement
with a previous work (5), where a polyclonal anti-Pol IV
serum was used for detecting Pol IV. Thus, Pol IV shows a
low undetectable expression level in P. aeruginosa cells.
DISCUSSION
According to our biochemical data, P. aeruginosa Pol IV
and MutS interacted with ␤ clamp. The contact between
the hydrophobic cleft of ␤ clamp and the conserved binding motif of Pol IV and MutS was the main anchoring interaction as indicated by the impaired interaction of the Pol
IV-5 and MutS␤ mutant versions. Confirming the importance of this contact, we showed that a peptide spanning
the CBM of MutS inhibited Pol IV–␤ clamp and MutS–
␤ clamp interactions. Notably, MutS was able to prevent
Pol IV binding to ␤ clamp, as well as to disrupt the Pol
IV–␤ clamp complex. These findings may be explained by
the facts that both MutS and Pol IV competed for binding to the protein-binding cleft of the ring, and the MutS–␤
clamp interaction exhibited a higher affinity than the Pol
IV–␤ clamp interaction. In our experiments we did not detect the formation of a ternary complex consisting of Pol IV,
MutS and ␤ clamp as it has been reported for other interacting partners (26,29,50–51). It is likely that MutS binding to ␤ clamp impedes the simultaneous association of Pol
IV by occupying both clefts, since MutS has four CBMs
per molecule, or by sterically inhibiting access of Pol IV to
the ring. In addition, we observed that the affinity of the
MutS–␤ clamp interaction increased in the presence of a
GT primed-DNA, which may be important for targeting
MutS to ␤ clamp molecules that are bound to a primed site
and close to a mismatch (see below the implications of this
assumption in the context of our proposed mechanism). It is
possible that a conformational change in MutS and ␤ clamp
brought about by binding to this DNA structure may have
promoted the interaction between these factors. The crystal
structures of ␤ clamp in complex with a primed-DNA and
MutS bound to mismatched bases differ from those in the
apo form, suggesting that binding to these DNA structures
induces a conformational change in both proteins (33,52).
Regulation of the association between binding partners and
␤ clamp by DNA structures has been documented. For example, the Pol III core (a complex formed by the ␣ polymerase, ⑀ exonuclease and ␪ subunits) develops a higher
affinity for ␤ clamp when the ring is placed on primedDNA, which assures a correct assembly of the replicative
Pol at a primed site to initiate processive synthesis (42). We
noticed striking effects of DNA structures on the ability of
MutS to regulate the Pol IV interaction with ␤ clamp. MutS
exerted a more strict control over Pol IV binding to ␤ clamp
in the presence of a primed-DNA containing or not a mismatch, may be ensuring that the Pol IV control function of
MutS is restricted to replication sites. In contrast, a singlestranded DNA substrate abolished the capability of MutS
for limiting the Pol IV–␤ clamp interaction. Single-stranded
DNA regions accumulate after DNA damage, which trig-
gers the SOS response by activating the co-protease activity
of RecA that facilitates autocleavage of LexA (48). Thus, it
is possible that MutS does not control Pol IV under these
stressful conditions wherein the mutagenic activity of Pol
IV is necessary to generate adaptive variants.
Our data also demonstrate that basal levels of Pol IV contribute to the normal chromosomal error rate in P. aeruginosa cells when MutS does not interact with the ␤ clamp
processivity factor. The following lines of evidence support this conclusion: (i) Pol IV deficiency significantly decreased AT > CG, CG > AT and AT > GC base substitution rates in the mutS␤ strain. In contrast, and similarly
to previous studies in E. coli (10), the mutation spectrum
was not modified by inactivation of the chromosomal dinB
gene in the WT strain: (ii) the mutS␤ strain showed a Pol IVdependent strand bias of mutation, which was not detected
in the WT strain: (iii) a higher proportion of base substitutions was present at sites involving the Pol IV-preferred
sequence GXC context in the mutS␤ strain compared to the
WT strain. At higher Pol IV levels, obtained by ectopic expression, this alternative Pol increased mutations on the P.
aeruginosa chromosome in a dose-dependent manner in the
mutS␤ strain, whereas such mutagenesis was limited in the
WT strain. This effect was not observed with the Pol IV-5
mutant, demonstrating the functional importance of the association with the processivity factor to target Pol IV to its
DNA substrate. On the other hand, the ability of Pol IV to
promote survival by bypassing ENU-induced DNA lesions
did not differ between the WT and mutS␤ strains containing
basal or higher Pol IV expression levels. Thus, the MutS–␤
clamp association limits Pol IV mutagenesis resulting from
the low fidelity replication of undamaged DNA, but it does
not affect accurate DNA synthesis by Pol IV as in TLS of
alkylating lesions.
In vitro studies have shown that Pol IV takes control of
the primer terminus from a stalled or, much less efficiently,
an actively replicating Pol III HE (comprised of Pol III
core, DnaX clamp loader and ␤ clamp) (26–27,29). Moreover, Pol IV is also able to recruit ␤ clamp from Pol III HE
replisomes, where the replicative Pol acts in concert with
the DnaB helicase and the DnaG primase (25,28). At Pol
IV/Pol III HE ratios similar to that found in exponentially
growing E. coli cells, the Pol switch proceeds through an intermediate in which Pol III HE and Pol IV bind to the same
␤ clamp without dissociation of the replicative Pol from the
ring (26,29). Pol III HE contains a high affinity CBM in the
␣ catalytic subunit and a weak CBM in the ⑀ exonuclease
subunit (50,51). Both sites in the ␤ dimer are occupied simultaneously by the CBMs of Pol III HE during processive
replication, whereas breakage of the ⑀–␤ clamp interaction
may occur during proofreading to transfer the primer template from ␣ to ⑀ (50,51). It has been suggested that Pol IV
is bound to ␤ clamp in an inactive state by contacting a secondary site and that it can switch with Pol III HE by gaining
access to the cleft that is weakly bound by ⑀ (26–27,50). Pol
III HE can remain bound to the ring by contacting the opposing cleft of ␤ clamp with the CBM of ␣ in an inactive
conformation while Pol IV is carrying out synthesis (26).
These data support a model in which Pol IV is able to associate with ␤ clamp at the replisome without interfering with
Pol III HE replication during normal growth, thereby al-
7710 Nucleic Acids Research, 2016, Vol. 44, No. 16
lowing a rapid exchange when Pol III HE is stalled (27,29).
However, the biological significance of this model remains
largely untested. It is not clear whether Pol IV is constitutively a part of the replisome. A recent work reported that
fluorescently tagged Pol IV localizes to the nucleoid in E.
coli cells under normal growth conditions (53). This localization differs from that of replisome components, which
form discrete foci (54), suggesting that this Pol may not be
associated with the replicative machinery, despite its high
basal concentration. Similarly, P. aeruginosa Pol IV may not
be associated with ␤ clamp resident in the replisome, since
it exhibited a low, undetectable expression level and a weak
affinity for the ring. Thus, Pol IV may be recruited to the
replication fork for switching with Pol III HE.
Within the framework of the present manuscript, and in
accordance with previously published findings, we propose
the following mechanism for the control of Pol IV–␤ clamp
interaction by MutS (Figure 8). Diagram A: Pol III HE has
control of the primed-DNA during normal replication by
occupying both clefts in the polymerization mode. Diagram
B: transient stalling of the replication machinery, owing to
inability to extend a terminal mismatch generated by Pol III
HE or encountering a lesion, can signal the recruitment of
Pol IV to the replisome. Pol IV gains access to the primer
terminus from Pol III HE by taking the cleft from ⑀ while
Pol III HE remains bound to the second cleft by the CBM
of ␣. Diagram C: if Pol IV activity generates a mismatch, by
introducing an incorrect nucleotide or extending a terminal
mismatch, MutS is able to be recruited since its affinity for ␤
clamp increases in the presence of the mispair. Thus, MutS
competes with Pol IV on ␤ clamp, leading to its dissociation
from the cleft. Diagram D: this could provide the opportunity for the ⑀ exonuclease to access the primer terminus,
resulting in the excision of the incorrect nucleotide. After
error removal, MutS loses affinity for ␤ clamp and leaves
the cleft on the ring. Pol III HE ultimately regains control
of the primed-site and resumes processive DNA synthesis.
Our model predicts that Pol IV can effectively act to alleviate stalling of the replication fork, but if its activity generates a mismatch, MutS can limit further replication by
Pol IV and impede the fixation of mutations. Alternatively,
MutS could also control the action of Pol IV associated with
␤ clamps that are not directly located at the replisome. It
has been shown that ␤ clamp is not immediately unloaded
between successive Okazaki fragments but, remains bound
to the newly synthesized DNA for a prolonged period of
time, resulting in the accumulation of ␤ clamp molecules
behind the fork (54,55). If MutS regulates binding of Pol IV
to these ␤ clamp molecules, the exonuclease activity of Pol
II could be involved in the removal of mispairs generated
by Pol IV. Consistent with their role in surveying the replication products of Pol IV, the ⑀ exonuclease and Pol II affect several Pol IV-dependent mutagenic phenomena (9,56).
In addition, it has been demonstrated that the eukaryotic
heterodimer Msh2/Msh6 suppress UV-induced mutagenesis by mediating the excision of incorrect nucleotides introduced by error-prone TLS Pols (57).
Of relevance to our model, we estimated that the concentration of MutS in exponentially growing P. aeruginosa
cells is ∼265 nM, a lower value than the KD (∼1 ␮M) of
the MutS–␤ clamp interaction. Since MutS binding to ␤
clamp significantly increased (∼3.5-fold) in the presence of
a primed-DNA containing a mispair, one could assume that
MutS only interacts with the processivity factor when a mismatch is generated during replication. Cytological studies in
exponentially growing bacteria have shown that MutS must
eventually contact ␤ clamp resident in the replisome. MutS
fused to a fluorescent protein colocalizes with the chromosome in all cells, but it forms foci in a subset of cells (18,58).
These foci are often coincident with foci associated with
replisome components, and their formation depends on the
MutS interaction with ␤ clamp (18). Accordingly, when a
fusion of the 57 C-terminal residues containing the CBM
from P. aeruginosa MutS to the Maltose Binding Protein
was affinity purified from PAO1 extracts, ␤ clamp and the
␣ catalytic subunit of Pol III HE co-eluted with the fusion as
identified by LC-MS/MS (data not shown). As we did not
detect interaction between MutS and ␣ in vitro (Supplementary Figure S4C), these data indicate that MutS may form
a complex with ␤ clamp and the ␣ subunit of Pol III.
How does MutS separate its function in the regulation of
Pol IV access to replication sites from that in the conserved
DNA MMR pathway? MutS is best known in the context
of MMR, removing bases mis-inserted during DNA replication. The repair process is initiated when MutS recognizes and binds to mismatches. Subsequently, mismatchbound MutS promotes loading of the other key MMR protein, MutL, onto DNA activating downstream steps that include discrimination of newly synthesized strand, excision
of the erroneous base and strand re-synthesis (59). MutL
coordinates MMR processes by interacting with MutS and
the majority of the proteins implicated in the subsequent
stages. Critical for correct MMR functioning is the interaction between MutS and MutL, which links mismatch recognition with the downstream events. We found that MutS interaction with MutL was inhibited when it formed a complex with ␤ clamp (Supplementary Figure S12A). In contrast, the MutS␤ mutant version interacted with MutL to
the same extent in the presence of ␤ clamp (Supplementary Figure S12B). Based on these data, we hypothesize
that the interaction of MutS with ␤ clamp favors its action
in the regulation of Pol IV access to replication sites and
limits MutS activity in repair processes. Thus, when mismatches generated by Pol IV activity are close to ␤ clamp,
they can be detected by MutS and removed according to our
proposed mechanism. These mispairs are not subjected to
MMR since the interaction of MutS with ␤ clamp inhibits
its association with MutL. When mismatches generated by
Pol IV (or replicative Pols) are far away from ␤ clamp, MutS
can interact with MutL and initiate the MMR pathway.
MutS from other bacteria also interacts with the processivity factor (13,18). This interaction plays a role in the
MMR of Bacillus subtilis by recruiting MutS to sites of
DNA replication and allowing for efficient mismatch detection (18,60), but not in the MMR of E. coli and P. aeruginosa (19,35), which raises questions about their functional
significance in both organisms. Our findings shed light on
the role of this interaction in P. aeruginosa and it is expected that the features described here can be extrapolated
to other bacteria. As in the case of gram-negative bacteria, a limited contribution of the MutS␣-PCNA association
with the MutS␣-dependent MMR pathway has been re-
Nucleic Acids Research, 2016, Vol. 44, No. 16 7711
Figure 8. Proposed model for the control of Pol IV–␤ clamp interaction by MutS. Our model illustrates the action on a single primer terminus, and it does
not depict other replisome components. Each of the CBMs is shown as balls. This model was proposed based on previous studies of the Pol III HE/Pol IV
switching (A and B), our findings (C) and a postulated action of the ⑀ exonuclease in the excision of mismatches generated by Pol IV (D). See ‘Discussion’
section for details regarding this model.
ported in eukaryotes (20,21). Importantly, MutS␣ regulates
Pol ␬- and Pol ␩-dependent TLS in human cells by promoting monoubiquitination of PCNA in response to UV and
oxidative damage, respectively (61,62). This transient posttranslational modification of PCNA facilitates recruitment
of these TLS Pols and their exchange with the replicative Pol
for lesion bypass. Hence, MutS may modulate the action of
alternative Pols in both bacteria and eukaryotes.
A high proportion of MutS-deficient cells of P. aeruginosa and other pathogenic bacteria often arise during longterm infections (63). Genome sequencing of P. aeruginosa
isolates taken from chronically infected patients with cystic
fibrosis revealed that the homopolymeric tracts of identical nucleotides are preferentially mutated in hypermutators
carrying an inactive mutS allele (64,65). These homopolymeric tracks are more common in genes related with the cell
envelope composition, suggesting that these mutations have
been selected to evade the host immune response. Thus,
this differential mutagenesis associated with the hypermutator phenotype may facilitate the establishment of longterm existence in the stressful conditions imposed by the
human host environment. The presence of homopolymeric
tracks in specific genes and the modulation of their mutation rates through inactivation of mutS have been shown to
be important for pathogenesis and host adaptation of several pathogens (66). Importantly, Pol IV strongly promotes
single nucleotide deletions within homopolymeric tracks
(5,11,43,67). It is tempting to speculate that Pol IV may be
involved in the mutagenesis of genes containing homopolymeric tracks in MutS-deficient cells, where Pol IV is not under the control of MutS, contributing to the pathoadaptation of pathogenic bacteria. Additionally, Pol IV-promoted
mutagenesis induced by sublethal levels of different antibiotics depends on the downregulation of MutS (12). Thus,
Pol IV mutagenic action under stressful conditions may
be determined by the inactivation or depletion of MutS,
which is consistent with our proposed model. Accordingly,
we found that Pol IV-dependent mutagenesis is not limited
by the MutS–␤ clamp interaction in P. aeruginosa cells expressing constitutively the SOS response to stress-induced
DNA damage. This may be explained by two ways: (i) MutS
expression is downregulated and (ii) the MutS action is regulated by other factors induced as part of the SOS response.
Additional studies will be necessary to address these possibilities.
In conclusion, our results reveal important insights into
a likely non-canonical function of the MMR protein MutS
in the regulation of a replication activity in bacteria involving complex protein–protein and protein–DNA interactions. These findings open up new interrogatives regarding the context and finer details of this mechanism.
7712 Nucleic Acids Research, 2016, Vol. 44, No. 16
SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online.
FUNDING
Consejo Nacional de Investigaciones Cientı́ficas y Técnicas
[PIP 20130100360]; Secretarı́a de Ciencia y Tecnologı́a de
la Universidad Nacional de Córdoba [30820110100360CB];
Agencia Nacional de Promoción Cientı́fica y Técnica [PICT
2011-1797]. Funding for open access charge: Consejo
Nacional de Investigaciones Cientı́ficas y Técnicas (PIP
20130100360).
Conflict of interest statement. None declared.
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